Pathogenic bacteria responsible for infectious diseases were once thought to be controllable through the use of a battery of antibiotics such as penicillin, streptomycin, tetracycline, and others. However, since the widespread use of antibiotics began in the 1950s, more and more bacteria have evolved to become resistant to one or more antibiotics. Multiple drug-resistant strains are increasingly common, particularly in hospitals.
Currently, nosocomial Staphylococcal infections exhibit multiple drug resistance. See, for example Archer et al., 1994, Antimicrob. Agents Chemother. 38:2231-2237. At this time, the remaining antibiotic that demonstrates the ability to kill most strains of Staphylococci is vancomycin. However, vancomycin resistant strains of both Staphylococcus and Enterococcus have already been isolated and reported by Zabransky et al., 1995, J. Clin. Microbiol. 33(4):791-793. Furthermore, transfer of resistance from Enterococci to Staphylococci has been previously documented by Woodford et al., 1995, J. Antimicrob. Chemother. 35:179-184. Streptococcus pneumoniae is a leading cause of morbidity and mortality in the United States (M.M.W.R., Feb. 16, 1996, Vol. 45, No. RR-1). Each year these bacteria cause 3,000 cases of meningitis, 50,000 cases of bacteremia, 500,000 cases of pneumonia, and 7,000,000 cases of otitis media. Case fatality rates are greater than 40% for bacteremia and greater than 55% for meningitis, despite antibiotic therapy. In the past, Streptococcus pneumoniae were uniformly susceptible to antibiotics; however, antibiotic resistant strains have emerged and are becoming widespread in some communities.
In addition, there are instances where antibiotic resistance is not an issue, yet a particular bacteria remains refractory to treatment using conventional antibiotics. Such is the case with Escherichia coli 0157:H7, a causative agent for food poisoning and death from undercooked meat. The Department of Agriculture estimates that 10 people die each day and another 14,000 become ill due to this bacteria. Unfortunately, conventional antibiotics are completely ineffective against this organism.
The history of antibiotic treatment of pathogenic bacteria is cyclical. Bacteria are remarkably adaptive organisms, and, for each new antibiotic that has been developed, resistant bacterial strains arise through the widespread use of the antibiotic. Thus, there is a constant need to produce new antibiotics to combat the next generation of antibiotic-resistant bacteria. Traditional methods of developing new antibiotics have slowed, and in the past two years only one new antibiotic has been approved by the FDA. Furthermore, according to Kristinsson (Microb. Drug Resistance 1(2):121 (1995)), "there are no new antimicrobial classes with activity against resistant Gram positives on the horizon."
Antisense therapy uses nucleic acids to specifically inhibit unwanted gene expression in cells. Antisense nucleic acids can be used to hybridize to and inhibit the function of an RNA, typically messenger RNA, by activating RNase H or physically blocking the binding of ribosomes or proteins, thus preventing translation of the mRNA. Antisense nucleic acids also include RNAs with catalytic activity (ribozymes), which can selectively bind to complementary sequences on a target RNA and physically destroy the target by mediating a cleavage reaction.
Antisense nucleic acids that bind to the DNA at the correct location can also prevent the DNA from being transcribed into RNA. These antisense nucleic acids are believed to bind to double-stranded DNA (forming triple-stranded DNA) and thereby inhibit gene expression.
Nucleic acids have also found use as aptamers in which their mode of action is the result of interactions with molecules other than DNAs or RNAs, e.g., proteins.
Nucleic acids have also been shown to stimulate the immune system in response to the presence of a CG motif (Yamamoto et al., 1994, Antisense Res. Devel. 4:119-122; Krieg et al., 1995, Nature 374:546-549). The mechanism of this stimulation is not clear but it does not seem to involve an antisense mechanism.
It has been demonstrated that the fate of internalized nucleic acids is critical to the success of nucleic acid therapy (Bennett, 1993, Antisense Res. Devel. 3:235-241). The rapid intracellular degradation of nucleic acids can be a barrier to their use. One of the major problems in utilizing naturally occurring phosphodiester nucleic acids is their rapid degradation by nucleases in mammalian cells or in serum-containing culture medium (Cohen, 1989, Oligodeoxynucleotides; Antisense Inhibitors of Gene Expression, Boca Raton, Fla., CRC Press). There is abundant evidence that modification of the backbone of nucleic acids confers varying degrees of nuclease resistance. Hoke et al., 1991, Nucl. Acids Res. 19:5743, compared phosphodiester backbone nucleic acids to fully modified phosphorothioate backbone nucleic acids and to chimeric phosphodiester and phosphorothioate backbone nucleic acids. Hoke et al. demonstrated that the phosphorothioate nucleic acids were degraded up to 45 times slower than the phosphodiester or chimeric backbone nucleic acids.
There have been reports that chimeric nucleic acids that are end-capped with nuclease resistant backbone linkages are resistant to degradation (Cohen, 1989, Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression, Boca Raton, Fla., CRC Press). However, Hoke et al. teaches that capped nucleic acids are rapidly degraded by intracellular endonucleases, and thus capping nucleic acids with nuclease resistant modifications may not be sufficient for sustaining pharmacological activities of nucleic acids in cells. Finally, Hoke et al. concludes that while capping of nucleic acids may provide protection from exonucleases in cell culture, the action of intracellular endonucleases is sufficient to degrade these capped nucleic acids when they enter a cell.
Another limitation on the therapeutic uses of nucleic acids has been their poor bioavailability. Oral bioavailability can be affected by acid degradation in the gut, enzymatic cleavage in the intestines, poor intestinal absorption and liver first pass effects (Hughes et al., 1995, Pharmaceutical Research 12:817). Crooke reported very limited (&lt;5%) bio-availability of nucleic acids in rodents (S. Crooke, 1997, in Antisense Nucleic Acid and Antisense RNA: Novel Pharmacological and Therapeutic Agents, B. Weiss ed., CRC Press Boca Raton, Fla., p. 17). The normal pH of the gastric hydrochloric acid (HCl) in the stomach is between 1 and 2 (A. Goth, 1974, Medical Pharmacology: Principles and Concepts, The C. V. Mosby Company, Saint Louis, Mo.). Nucleic acids are sensitive to acid depurination and cleavage of the DNA or RNA backbone. Exposure of nucleic acids for as short a time as 10 minutes at room temperature at a pH of 1-2 will cause depurination.
The sole effort to use nucleic acids as antibiotics to date has involved their use as antisense molecules targeted to hybridize and inhibit expression of specific bacterial genes, thereby inhibiting bacterial growth. See Lupski et al., U.S. Pat. No. 5,294,533 ('533 patent) and PCT publication NO. WO 96/29399. Although this method can be effective, it is limited in its scope of use, the strength of the effect, and the use of an antisense molecule is limited to a particular targeted organism or closely related organisms.
There is a need for a new class of broad-spectrum antibiotic agents that is effective against a wide range of bacteria, including bacteria resistant to many conventional antibiotics. In addition, there is a need for such a broad-spectrum antibiotic that is non-toxic to the treated host.